Genetic Adversity Among Walnut Varieties With ISSR And SSR

In this study, ISSR and SSR molecular marker technique were used to analyse thegenetic diversity and relationship of18walnut samples from North West Agricultural andForestry University experiment stations,to reveal the difference of genetic diversity and laythe foundation for further breeding.18walnut samples tested from Liaoning, Shaanxi,Xinjiang, Shandong, Hebei and United States include15different varieties walnut and3seedling good strains.The main contents and results of this experiment are as follows:1.Genome DNA extraction: Taking young leaves as samplesï¼› modified CTAB wasused to extract genome DNA which was tested by1%agarose gel electrophoresis andSpectrophotometer.With RNA enzyme purifying, the production of DNA although decreased,but the purity was significantly improved.2.Establish the optimal reaction system，ISSR reaction system20μL total reactionsystem,2mmol/L Mg²⁺,2U Taq enzyme,0.4mmol/L dNTP,1.5ng/μL template, and0.6μmol/L primer. The amplification program: first94℃pre-denaturation5min thendenaturation94℃30s,45℃annealing30s72℃extension60s for32cyclesï¼› at last72°C extension for10min,4℃preservation.SSR reaction system:20μL total reaction system,4mmol/L Mg²⁺, and0.5U Taqenzyme,0.4mmol/L dNTP,6ng/μL template, and0.8μ mol/L primer. The amplificationprogram: first94℃pre-denaturation3min then94℃denaturation for30s,45°Cannealing30s,72℃extension45s for30cyclesï¼› at last72℃extension for5min,4°Cpreservation.3primer screening:15ISSR primers and12pairs of SSR primers which amplified clearand sTab. polymorphic were screened.4Genetic diversity: ISSR molecular marker detected a total of187bands, of which132presented polymorphism. Polymorphism percentage of ISSR was70.59%. The similaritycoefficient ranged0.2500⁰.7639. Polymorphic information content （PIC） ranged from0.7105to0.9151, and each value were greater than0.5, indicating that15primers sitesshowed a high degree of polymorphism. SSR molecular marker detected a total of174bands,of which118presented polymorphism. Polymorphism percentage of SSR was67.82%. Thesimilarity coefficient ranged0.1594⁰.8036. Polymorphic information content （PIC） ranged0.7422⁰.8962, and each value were greater than0.5, indicating that12primers sites showed a high degree of polymorphism,also showed that the genetic resources of these samples arevery rich,can be used as breeding source.5. Cluster analysis: Integrated bands ISSR and SSR amplified to cluster analysis,18materials can be divided into four groups: the first group, Xiluo3, Qingxiang, Xilin3,Liaoning4, Xinjiang2,Xiangling, Xilin2, S1, S2, S3, Turair,Wen185ï¼›second group, Wina,Chicoï¼› third group, Qinglin, W06-1,Lvxiangï¼›fourth group: Yihe1.6. ISSR and SSR analysis: Mantel test was used to analyse correlation of ISSR and SSR（r=0.6257, P=1）, indicating that ISSR markers and SSR markers in this study is not fullyconsistent, this is related to the principle.